Alternative Substrates for Estimating TCE-degrading Capabilities of Toluene-oxidizing Bacteria

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Title: Alternative Substrates for Estimating TCE-degrading Capabilities of Toluene-oxidizing Bacteria
Author: Hicks, Kristin Adair
Advisors: Dean Hesterberg, Committee Member
Deanna Osmond, Committee Co-Chair
Michael R. Hyman, Committee Co-Chair
Abstract: One of the primary impediments to the implementation of bioremediation is uncertainty about success in the field. Soils and microbial populations are heterogeneous and it is difficult to extrapolate biodegradation rates from small samples to field scale. While biodegradation rates can be estimated from microcosm studies, in situ methods offer a more meaningful gauge of resident microbial activity. One method used to estimate biodegradation rates in the field is the newly developed Push-Pull technique. While this technique can be conducted on site, it is normally not possible to use target pollutants as the reactive substrates. Consequently, alternative, benign reactive tracers must be used. Ideally, these alternative, reactive tracers interrogate the same enzyme systems that are responsible for the biodegradation of the target pollutant. The objective of this study was to develop a reactive tracer system that could be used to assess toluene-dependent trichloroethylene (TCE) degradation. Our approach has been to determine whether a series of pure strains of toluene-oxidizing bacteria (Burkholderia cepacia G4, Pseudomonas putida F1, Pseudomonas putida mt2, Pseudomonas mendocina KR1), each with different toluene-oxidizing enzymes systems, are capable of cometabolically oxidizing a series of eleven potential alternative substrates. These substrates include simple alkenes, alkanes, and cyclic alkanes. The kinetics (Ks and Vmax) of the biotransformation of these compounds have been determined. While oxidation products were observed for a number of these substrates in connection with one or more of the test organisms, isobutylene was co-oxidized by all test organisms. Oxidation of isobutylene by each organism yielded kinetics constants comparable to the corresponding kinetics of TCE degradation. The enzyme system expressed by Burkholderia cepacia G4 catalyzed the epoxidation of isobutylene while the remaining enzyme systems catalyzed allylic alcohol formation. Isobutylene has potential in field scale Push-Pull studies as a tool for evaluating rates of aerobic toluene-dependent TCE degradation and of differentiating the relative contributions of the TCE-degrading population. A pilot study of this alternative substrate at Edwards Air Force Base will test whether it can be used successfully to estimate in situ degradation of TCE. Analysis of isobutylene oxidation products in toluene-enriched ground water may offer an inexpensive and effective method of measuring the degradation of TCE at contaminated sites nationwide.
Date: 2002-07-24
Degree: MS
Discipline: Soil Science

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